Von Hippel Lindau Disease (VHL) is a devastating disease associated with various forms of cancer in multiple organ systems frequently leading to serious clinical complications and death. Using DNA linkage analysis, we have recently provided conclusive evidence that the gene causing VHL is located on the short arm of chromosome 3 in the neighborhood of the c- rafI oncogene. Consequently, we propose to bracket, and isolate the disease gene based on the knowledge of its chromosomal location. The currently available DNA markers together with new markers generated from chromosome 3 specific libraries, will be used to define loci that are more tightly linked to, and flank the VHL defect. This should lead to the development of a much needed diagnostic test, and will provide the basis for the isolation of the defective gene itself. In order to gain insights into the mechanism of tumorigenesis in VHL, chromosome 3p markers will be applied to search for chromosomal deletions in VHL tumors. The deleted regions in VHL tumors will be compared with the deletions in the sporadic counterparts of VHL tumors, e.g. in renal cell carcinoma, to address the question of whether sporadic and familial forms of VHL-associated tumor types result from similar pathogenetic mechanisms affecting the same gene locus. Overlapping DNA clones representing the minimum genetically definable region containing the VHL gene will be isolated using one or a combination of cloning strategies. The genes encoded in this region will be analyzed at the DNA, RNA, and protein level. An in depth comparison of the genes will be performed between VHL tumors and corresponding normal tissue, and between tissue from individuals with and without VHL to identify the gene causing VHL. The isolation of the VHL defect might provide the basis for the development of rational therapies in VHL, and could have important impacts not only for VHL patients, but for a much larger number of cancer patients suffering from the sporadic counterparts of VHL tumors, including renal cell carcinoma. Ultimately, the isolation and characterization of the VHL defect should yield new insights into the function of the normal counterpart of the VHL gene which might play a fundamental role in growth and differentiation of endothelial cells in the human central nervous system and other organ systems.